Oxygen homeostasis in mammals is tightly regulated, necessitated by the need to maintain sufficient levels for critical oxygen-dependent processes while minimizing the production of oxygen reactive species that are capable of causing oxidative damage to DNA, lipids, and proteins. In a state of hypoxia, where oxygen demand exceeds supply, a physiological response is mounted that increases the capacity of blood to carry oxygen to tissues and alters cellular metabolism, such as facilitating ATP production by anaerobic glycolysis. The hypoxia-inducible factors (HIFs) are key transcriptional regulators of this hypoxic response. These factors have also been implicated in the pathology of many major human diseases, including cancer, myocardial infarction, ischemia and preeclampsia (Harris, Nat. Rev. Cancer, 2002, 2, 38-47); (Lee et al., N Engl J Med, 2000, 342, 626-633); (Aplin, J Clin Invest, 2000, 105, 559-560)). Cells are typically cultured in the laboratory at an ambient oxygen concentration of 21%, but cells in the human body are exposed to much lower oxygen concentrations ranging from 16% in the lungs to less than 6% in most other organs of the body and often significantly less in tumors (Semenza, Trends Mol Med, 2001, 7, 345-350).
The HIF proteins are heterodimers consisting of HIF1-beta and one of three alpha subunits, HIF1-alpha, HIF2-alpha and HIF3-alpha (Safran and Kaelin, J Clin. Invest., 2003, 111, 779-783). The discovery of the HIF proteins was enabled by the identification of a minimal hypoxia-responsive element (HRE) in the 3′ enhancer of the erythropoietin gene (Wang and Semenza, Proc Natl Acad Sci USA, 1993, 90, 4304-4308). Subsequent analysis identified the HIF protein as a phosphorylation-dependent protein that binds DNA under hypoxic conditions (Wang and Semenza, J Biol Chem, 1993, 268, 21513-21518). Purification of this DNA-binding factor revealed HIF was a heterodimeric complex consisting of a novel protein, HIF1-alpha, and the aryl hydrocarbon nuclear translocator (ARNT, also termed HIF1-beta), previously identified as a binding partner of the dioxin/aryl hydrocarbon receptor (Wang and Semenza, J Biol. Chem., 1995, 270, 1230-1237.); (Hoffman et al., Science, 1991, 252, 954-958). HIF proteins belong to a class of transcription factors termed basic helix-loop-helix proteins, grouped by two conserved domains. The basic region consists of approximately 15 predominantly basic amino acids responsible for direct DNA binding. This region is adjacent to two amphipathic alpha helices, separated by a loop of variable length, which forms the primary dimerization interface between family members (Moore et al., Proc Natl Acad Sci USA, 2000, 97, 10436-10441).
HIF1-beta is a key player in two major signaling pathways, the hypoxic-response pathway and the aryl hydrocarbon receptor (AHR) pathway. Since the discovery of HIF1-alpha/HIF1-beta involvement in erythropoietin transcription, HIF activity has been detected in various non-erythropoietin-producing cell lines cultured under hypoxic conditions (Wang and Semenza, Proc Natl Acad Sci USA, 1993, 90, 4304-4308); (Maxwell et al., Proc Natl Acad Sci USA, 1993, 90, 2423-2427), providing the first evidence that the HIF1 dimer not only activates the erythropoietin gene, but is part of a widespread oxygen-sensing and signal transduction mechanism. Under normoxic conditions, HIF1-alpha is rapidly degraded due to the oxygen-dependent hydroxylation of specific proline residues that mark the protein for proteasomal degradation (Jewell et al., Faseb J, 2001, 15, 1312-1314); (Gorlach et al., Biochim Biophys Acta, 2000, 1493, 125-134). Under hypoxic conditions, this hydroxylation is reversed, and the protein is further stabilized by phosphorylation (Wang et al., Biochem Biophys Res Commun, 1995, 216, 669-675). Subsequently, the protein is translocated to the nucleus, where it interacts with HIF1-beta to form a heterodimeric transcription factor (Kallio et al., Embo J, 1998, 17, 6573-6586). Studies in HIF1-beta deficient cells revealed an absolute requirement for this dimerization step for the transcriptional activation of hypoxia response element genes (Wood et al., J Biol Chem, 1996, 271, 15117-15123). Categories of genes that are activated by the HIF1 dimer include oxygen transport genes, such as erythropoietin (Semenza et al., J Biol Chem, 1994, 269, 23757-23763) and transferrin (Rolfs et al., J Biol Chem, 1997, 272, 20055-20062); genes involved in angiogenesis, such as VEGF (Levy et al., J Biol Chem, 1995, 270, 13333-13340); and genes involved in anaerobic metabolism, such as glucose transporter 1 (Ebert et al., J Biol Chem, 1995, 270, 29083-29089). Hypoxia-induced genes such as VEGF are thought to play a role in promoting angiogenesis and subsequent tumor growth (Harris, Nat. Rev. Cancer, 2002, 2, 38-47).
HIF transcriptional activity is precisely regulated by cellular oxygen concentration. Whereas changes in oxygen levels do no affect HIF1-beta protein levels, the abundance of the HIF-alpha subunits is markedly increased upon exposure of cells to hypoxia, primarily due to stabilization of the alpha subunits (Safran and Kaelin, J. Clin. Invest., 2003, 111, 779-783). HIF2-alpha mRNA and protein is expressed at low levels in tissue culture cells, but protein expression is markedly induced by exposure to 1% oxygen, a hypoxic state (Wiesener et al., Blood, 1998, 92, 2260-2268). The HIF2-alpha/HIF1-beta heterodimer protein binds to the hypoxic response element, which contains the core recognition sequence 5′-TACGTG-3′ and is found in the cis-regulatory regions of hypoxia-regulated genes (Ema et al., Proc. Natl. Acad. Sci. U.S.A., 1997, 94, 4273-4278); (Hogenesch et al., J. Biol. Chem., 1997, 272, 8581-8593). Binding of the heterodimer to the HRE induces gene expression (Wiesener et al., Blood, 1998, 92, 2260-2268).
In contrast to the HIF-alpha subunits, HIF1beta is stable under both hypoxic and normoxic conditions, and also participates in the aryl hydrocarbon receptor (AHR) signaling pathway. AHR is a cytoplasmic receptor protein that translocates to the nucleus after ligand binding. Ligands of AHR include 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), an environmental toxin that is a by-product of industrial processes (Poland et al., J Biol Chem, 1976, 251, 4936-4946); polycylic aromatic hydrocarbons, found in cigarette smoke and smog (Reisz-Porszasz et al., Mol Cell Biol, 1994, 14, 6075-6086); and heterocyclic amines, found in some cooked meats (Reisz-Porszasz et al., Mol Cell Biol, 1994, 14, 6075-6086). After ligand-binding and nuclear translocation, AHR forms a dimer with HIF1-beta, resulting in the activation of a number of genes involved in drug metabolism, such as the cytochromes P450, CYP1A1, CYP1A2, and CYP1B1. AHR/HIF1-beta dimers are capable of activating a range of other genes regulated by the dioxin response element (DRE), resulting in some of the toxic and carcinogenic effects associated with many of the AHR ligands, such as immunotoxicity, developmental and reproductive toxicity, disruption of endocrine pathways, a wasting syndrome, and tumor promotion (Safe, Toxicol Lett, 2001, 120, 1-7). Ohtake and colleagues (Ohtake et al., Nature, 2003, 423, 545-550) demonstrated that the AHR/HIF1-beta heterodimer directly associates with the estrogen receptors ER-alpha and ER-beta. They showed that this association results in the recruitment of unliganded estrogen receptor and coactivator p300 to estrogen-responsive gene promoters, leading to activation of transcription and estrogenic effects and giving rise to the adverse estrogen-related actions of dioxin-type environmental contaminants.
The role of HIF1-beta in both hypoxia-induced and AHR signaling pathways makes it an attractive therapeutic candidate, as both of these pathways have been linked to various forms of malignancies (Harris, Nat. Rev. Cancer, 2002, 2, 38-47); (Safe, Toxicol Lett, 2001, 120, 1-7). The angiogenic promoting capabilities of HIF1-beta also mark this gene as a potential therapeutic target for a variety of angiogenic disorders, such as arthritis, cardiovascular diseases, skin conditions, aberrant wound healing and ocular conditions (e.g., macular degeneration, diabetic retinopathy, diabetic macular edema and retinopathy of prematurity).
PCT publication WO 02/053735 discloses the use of an oligonucleotide 35 nucleotides in length as a PCR primer for amplification of the HIF1-beta sequence.
U.S. Pat. No. 6,352,829 discloses the use of an oligonucleotide 26 nucleotides in length as a PCR primer for amplification of the HIF1-beta sequence.
U.S. pre-grant publication 2004-0152655 discloses antisense oligonucleotide compounds for inhibiting HIF1-alpha.
U.S. pre-grant publication 2004-0096848 discloses oligomeric compounds directed against HIF1-alpha.
U.S. pre-grant publication 2005-0163781 discloses compounds for use as inhibitors of hypoxia-induced genes, such as HIF1-alpha and HIF2-alpha, to treat adhesion formation.
U.S. pre-grant publication 2004-0180357 discloses HIF1-alpha siRNA compounds for downregulating expression of HIF1-alpha and VEGF and inhibiting angiogenesis.
U.S. pre-grant publication 2005-0148496 discloses methods of treating inflammatory disorders such as rheumatoid arthritis using compounds that inhibit HIF1-alpha activity.
U.S. pre-grant publication 2004-0086498 discloses methods for treating animals with advanced or large tumor burdens by administration of an immunotherapeutic agent and a tumor growth restricting agent, such as an expression vector encoding an antisense version of HIF1-alpha.
U.S. pre-grant publication 2005-0070474 discloses methods of treating tumors using an agent to increase B7-H3 in combination with an agent to inhibit HIF1-alpha, HIF2-alpha or HIF3-alpha.
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of HIF1-beta and to date, investigative strategies aimed at modulating the function of HIF1-beta have involved the use of antibodies and inactive mutants. Consequently, there remains a long felt need for additional agents capable of effectively inhibiting HIF1-beta function.
Antisense technology is an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of HIF1-beta expression. Provided herein are antisense compounds for inhibition of HIF1-beta expression. The disclosed compounds can used for treating or preventing conditions associated with HIF1-beta, such as cancer and angiogenic disorders.